scholarly journals Inositol 1,2,3-trisphosphate and inositol 1,2- and/or 2,3-bisphosphate are normal constituents of mammalian cells

1995 ◽  
Vol 306 (2) ◽  
pp. 557-564 ◽  
Author(s):  
C J Barker ◽  
P J French ◽  
A J Moore ◽  
T Nilsson ◽  
P O Berggren ◽  
...  
Keyword(s):  
Beta Cells ◽  
Mammalian Cells ◽  
Lymphoid Cells ◽  
Inositol Polyphosphate ◽  
Monocytic Differentiation ◽  
Isotopic Equilibrium ◽  
Inositol Polyphosphates ◽  
Hl60 Cells ◽  
Cellular Iron ◽  
In Cells

1. An inositol trisphosphate (InsP3) distinct from Ins(1,4,5)P3 and Ins(1,3,4)P3, which we previously observed in myeloid and lymphoid cells [French, Bunce, Stephens, Lord, McConnell, Brown, Creba and Michell (1991) Proc R. Soc. London B 245, 193-201; Bunce, French, Allen, Mountford, Moore, Greaves, Michell and Brown (1993) Biochem. J. 289, 667-673], is present in WRK1 rat mammary tumour cells and pancreatic endocrine beta-cells. 2. It has been identified as Ins(1,2,3)P3 by a combination of oxidation to ribitol, a structurally diagnostic polyol, and ammoniacal hydrolysis to identified inositol monophosphates. 3. Ins(1,2,3)P3 concentration in HL60 cells changed little during stimulation by ATP or fMetLeuPhe or during neutrophilic or monocytic differentiation, and Ins(1,2,3)P3 was unresponsive to vasopressin in WRK1 cells. 4. Ins(1,2,3)P3 was usually more abundant than Ins(1,4,5)P3, often being present at concentrations between approximately 1 microM and approximately 10 microM. 5. HL60, WRK-1 and lymphoid cells also contain Ins(1,2)P2 or Ins(2,3)P2, or a mixture of these two enantiomers, as a major InsP2 species. 6. Ins(1,2,3)P3 and Ins(1,2)P2/Ins(2,3)P2 are readily detected in cells labelled for long periods, but not in acutely labelled cells. This behaviour resembles that of InsP6, the most abundant cellular inositol polyphosphate that includes the 1,2,3-trisphosphate motif, which also achieves isotopic equilibrium with inositol only slowly. 7. Ins(1,2,3)P3 is the major InsP3 that accumulates during metabolism of InsP6 by WRK-1 cell homogenates. 8. Possible metabolic relationships between Ins(1,2,3)P3, Ins(1,2)P2/Ins(2,3)P2 and other inositol polyphosphates in cells, and a possible role for Ins(1,2,3)P3 in cellular iron handling, are considered.

scholarly journals An ATP-responsive metabolic cassette comprised of inositol tris/tetrakisphosphate kinase 1 (ITPK1) and inositol pentakisphosphate 2-kinase (IPK1) buffers diphosphosphoinositol phosphate levels

2020 ◽  
Vol 477 (14) ◽  
pp. 2621-2638 ◽  
Author(s):  
Hayley Whitfield ◽  
Gaye White ◽  
Colleen Sprigg ◽  
Andrew M. Riley ◽  
Barry V.L. Potter ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Inositol Phosphate ◽  
High Energy ◽  
Dependent Manner ◽  
Inositol Polyphosphate ◽  
Inositol Polyphosphates ◽  
Inositol Pyrophosphate ◽  
Relative Contribution ◽  
Chromatographic Resolution ◽  
Molecular Signals

Inositol polyphosphates are ubiquitous molecular signals in metazoans, as are their pyrophosphorylated derivatives that bear a so-called ‘high-energy’ phosphoanhydride bond. A structural rationale is provided for the ability of Arabidopsis inositol tris/tetrakisphosphate kinase 1 to discriminate between symmetric and enantiomeric substrates in the production of diverse symmetric and asymmetric myo-inositol phosphate and diphospho-myo-inositol phosphate (inositol pyrophosphate) products. Simple tools are applied to chromatographic resolution and detection of known and novel diphosphoinositol phosphates without resort to radiolabeling approaches. It is shown that inositol tris/tetrakisphosphate kinase 1 and inositol pentakisphosphate 2-kinase comprise a reversible metabolic cassette converting Ins(3,4,5,6)P4 into 5-InsP7 and back in a nucleotide-dependent manner. Thus, inositol tris/tetrakisphosphate kinase 1 is a nexus of bioenergetics status and inositol polyphosphate/diphosphoinositol phosphate metabolism. As such, it commands a role in plants that evolution has assigned to a different class of enzyme in mammalian cells. The findings and the methods described will enable a full appraisal of the role of diphosphoinositol phosphates in plants and particularly the relative contribution of reversible inositol phosphate hydroxykinase and inositol phosphate phosphokinase activities to plant physiology.

scholarly journals Phospholipase C of Cryptococcus neoformans Regulates Homeostasis and Virulence by Providing Inositol Trisphosphate as a Substrate for Arg1 Kinase

2013 ◽  
Vol 81 (4) ◽  
pp. 1245-1255 ◽  
Author(s):  
Sophie Lev ◽  
Desmarini Desmarini ◽  
Cecilia Li ◽  
Methee Chayakulkeeree ◽  
Ana Traven ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Phospholipase C ◽  
Mammalian Cells ◽  
Galleria Mellonella ◽  
Inositol Trisphosphate ◽  
Infection Model ◽  
Inositol Polyphosphate ◽  
Inositol Polyphosphates ◽  
Content Type ◽  
Capsule Production

ABSTRACTPhospholipase C (PLC) ofCryptococcus neoformans(CnPlc1) is crucial for virulence of this fungal pathogen. To investigate the mechanism of CnPlc1-mediated signaling, we established that phosphatidylinositol 4,5-bisphosphate (PIP2) is a major CnPlc1 substrate, which is hydrolyzed to produce inositol trisphosphate (IP3). InSaccharomyces cerevisiae, Plc1-derived IP3is a substrate for the inositol polyphosphate kinase Arg82, which converts IP3to more complex inositol polyphosphates. In this study, we show that inC. neoformans, the enzyme encoded byARG1is the major IP3kinase, and we further demonstrate that catalytic activity of Arg1 is essential for cellular homeostasis and virulence in theGalleria mellonellainfection model. IP3content was reduced in the CnΔplc1mutant and markedly increased in the CnΔarg1mutant, while PIP2was increased in both mutants. The CnΔplc1and CnΔarg1mutants shared significant phenotypic similarity, including impaired thermotolerance, compromised cell walls, reduced capsule production and melanization, defective cell separation, and the inability to form mating filaments. In contrast to theS. cerevisiae ARG82deletion mutant (ScΔarg82) strain, the CnΔarg1mutant exhibited dramatically enlarged vacuoles indicative of excessive vacuolar fusion. In mammalian cells, PLC-derived IP3causes Ca2+release and calcineurin activation. Our data show that, unlike mammalian PLCs, CnPlc1 does not contribute significantly to calcineurin activation. Collectively, our findings provide the first evidence that the inositol polyphosphate anabolic pathway is essential for virulence ofC. neoformansand further show that production of IP3as a precursor for synthesis of more complex inositol polyphosphates is the key biochemical function of CnPlc1.

scholarly journals A Proteomic Analysis of the Effects of Yessotoxin and Di-Desulfoyessotoxin on Yeast and Mammalian Cells

2021 ◽  
Author(s):  
◽  
Sarah Cordiner
Keyword(s):  
Cell Morphology ◽  
Hepg2 Cells ◽  
Mammalian Cells ◽  
Live Cells ◽  
Shellfish Poisoning ◽  
Hl60 Cells ◽  
Apoptosis Inducer ◽  
Blood Leukocyte ◽  
Shock Proteins

<p>Yessotoxin (YTX) is a disulfated polycyclic polyether, produced by dinoflagellate algae. It is known to accumulate in edible shellfish, raising concerns about its potential risk to human health. YTX was initially classified as a diarrhetic shellfish poisoning toxin, due to commonly being extracted alongside toxins of this variety. However, YTX does not induce any of the effects characteristic of this group. A separate category for YTXs was established by the European Commission in 2002 and a limit of 1 mg/kg of shellfish meat was established. YTX has been shown to be an apoptosis inducer in a variety of cell lines in vitro. It has also been shown to be lethal to mice when administered by intra-peritoneal injection. However, when administered orally only limited toxicity is observed. The di-desulfated derivative (dsYTX) has also been shown to be lethal to mice following intra-peritoneal injection. However it causes damage mainly to the liver, whereas YTX appears to target the heart. The mechanism of action of YTX is still unknown. The goals of this project were to use proteomic techniques, to examine the effects of YTX and dsYTX on Saccharomyces cerevisiae and human promyelocytic leukemic blood leukocyte (HL60) cells. Young et al. (2009) showed that the major proteins affected by YTX in HepG2 cells were heterogeneous ribonucleoproteins (hnRNPs), lamins, cathepsins and heat shock proteins. HnRNPs had not previously been identified as possible targets of YTX. Alterations of hnRNP levels were also seen in HL60 cells treated with microtubule stabilising agents, peloruside A or paclitaxel (Wilmes et al., 2011, 2012). No differences in cell morphology or significant changes in protein abundance were observed when S. cerevisiae cells were exposed to YTX. A small number of significant changes in abundance were detected when these cells were exposed to dsYTX. The small number of protein changes seen is possibly due to poor toxin entrance into the cell through the yeast cell wall, lack of protein targets structurally homologous to those found in mammalian cells, or fast removal of the toxin through export pumps. Twenty-four hour incubation of HL60 cells with YTX resulted in increased cell death but no change in cell morphology. Treatment with dsYTX caused cells to aggregate into clusters and a 24% decrease in the number of live cells. Increases were found in the abundance of β-actin, hnRNP A and BiP proteins in response to dsYTX treatment. Decreases in these proteins were seen in HepG2 cells treated with YTX for 24 hours. As seen in S. cerevisiae cells, dsYTX had a greater effect in HL60 cells compared with YTX. Overall, the results provide some support for the previously identified effect on hnRNPs in mammalian cells exposed to YTX.</p>

scholarly journals A Proteomic Analysis of the Effects of Yessotoxin and Di-Desulfoyessotoxin on Yeast and Mammalian Cells

2021 ◽  
Author(s):  
◽  
Sarah Cordiner
Keyword(s):  
Cell Morphology ◽  
Hepg2 Cells ◽  
Mammalian Cells ◽  
Live Cells ◽  
Shellfish Poisoning ◽  
Hl60 Cells ◽  
Apoptosis Inducer ◽  
Blood Leukocyte ◽  
Shock Proteins

<p>Yessotoxin (YTX) is a disulfated polycyclic polyether, produced by dinoflagellate algae. It is known to accumulate in edible shellfish, raising concerns about its potential risk to human health. YTX was initially classified as a diarrhetic shellfish poisoning toxin, due to commonly being extracted alongside toxins of this variety. However, YTX does not induce any of the effects characteristic of this group. A separate category for YTXs was established by the European Commission in 2002 and a limit of 1 mg/kg of shellfish meat was established. YTX has been shown to be an apoptosis inducer in a variety of cell lines in vitro. It has also been shown to be lethal to mice when administered by intra-peritoneal injection. However, when administered orally only limited toxicity is observed. The di-desulfated derivative (dsYTX) has also been shown to be lethal to mice following intra-peritoneal injection. However it causes damage mainly to the liver, whereas YTX appears to target the heart. The mechanism of action of YTX is still unknown. The goals of this project were to use proteomic techniques, to examine the effects of YTX and dsYTX on Saccharomyces cerevisiae and human promyelocytic leukemic blood leukocyte (HL60) cells. Young et al. (2009) showed that the major proteins affected by YTX in HepG2 cells were heterogeneous ribonucleoproteins (hnRNPs), lamins, cathepsins and heat shock proteins. HnRNPs had not previously been identified as possible targets of YTX. Alterations of hnRNP levels were also seen in HL60 cells treated with microtubule stabilising agents, peloruside A or paclitaxel (Wilmes et al., 2011, 2012). No differences in cell morphology or significant changes in protein abundance were observed when S. cerevisiae cells were exposed to YTX. A small number of significant changes in abundance were detected when these cells were exposed to dsYTX. The small number of protein changes seen is possibly due to poor toxin entrance into the cell through the yeast cell wall, lack of protein targets structurally homologous to those found in mammalian cells, or fast removal of the toxin through export pumps. Twenty-four hour incubation of HL60 cells with YTX resulted in increased cell death but no change in cell morphology. Treatment with dsYTX caused cells to aggregate into clusters and a 24% decrease in the number of live cells. Increases were found in the abundance of β-actin, hnRNP A and BiP proteins in response to dsYTX treatment. Decreases in these proteins were seen in HepG2 cells treated with YTX for 24 hours. As seen in S. cerevisiae cells, dsYTX had a greater effect in HL60 cells compared with YTX. Overall, the results provide some support for the previously identified effect on hnRNPs in mammalian cells exposed to YTX.</p>

scholarly journals Selection of down-regulated sequences along the monocytic differentiation of leukemic HL60 cells

FEBS Letters ◽  
1997 ◽  
Vol 414 (1) ◽  
pp. 146-152 ◽  
Author(s):  
S Herblot ◽  
A Vekris ◽  
A Rouzaut ◽  
F Najeme ◽  
C de Miguel ◽  
...  
Keyword(s):  
Monocytic Differentiation ◽  
Hl60 Cells ◽  
Selection Of

scholarly journals Methylation of deoxyribonucleic acid in cultured mammalian cells by N-methyl-N′-nitro-N-nitrosoguanidine. The influence of cellular thiol concentrations on the extent of methylation and the 6-oxygen atom of guanine as a site of methylation

1970 ◽  
Vol 116 (4) ◽  
pp. 693-707 ◽  
Author(s):  
P. D. Lawley ◽  
Carolyn J. Thatcher
Keyword(s):  
Oxygen Atom ◽  
Nucleic Acids ◽  
Mammalian Cells ◽  
Dimethyl Sulphate ◽  
Biological Effects ◽  
Methylation Of Dna ◽  
Thiol Content ◽  
Cellular Thiol ◽  
In Cells

1. In neutral aqueous solution N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) yields salts of nitrocyanamide as u.v.-absorbing products. With cysteine, as found independently by Schulz & McCalla (1969), the principal product is 2-nitràminothiazoline-4-carboxylic acid. Both these reactions liberate the methylating species; thiols enhance the rate markedly at neutral pH values. An alternative reaction with thiols gives cystine, presumably via the unstable S-nitrosocysteine. 2. Thiols (glutathione or N-acetylcysteine) in vitro at about the concentration found in mammalian cells enhance the rate of methylation of DNA markedly over that in neutral solution. 3. Treatment of cultured mammalian cells with MNNG results in rapid methylation of nucleic acids, the extent being greater the higher the thiol content of the cells. Rodent embryo cells are more extensively methylated than mouse L-cells of the same thiol content. Cellular thiol concentrations are decreased by MNNG. Proteins are less methylated by MNNG than are nucleic acids. 4. Methylation of cells by dimethyl sulphate does not depend on cellular thiol content and protein is not less methylated than nucleic acids. Methylation by MNNG may therefore be thiol-stimulated in cells. 5. Both in vitro and in cells about 7% of the methylation of DNA by MNNG occurs at the 6-oxygen atom of guanine. The major products 7-methylguanine and 3-methyladenine are given by both MNNG and dimethyl sulphate, but dimethyl sulphate does not yield O6-methylguanine. Possible reaction mechanisms to account for this difference between these methylating agents and its possible significance as a determinant of their biological effects are discussed.

scholarly journals Diffusion in the aqueous compartment.

1984 ◽  
Vol 99 (1) ◽  
pp. 180s-187s ◽  
Author(s):  
A M Mastro ◽  
A D Keith
Keyword(s):  
Rotational Motion ◽  
Spin Label ◽  
Mammalian Cells ◽  
Translational Motion ◽  
Diffusion Constant ◽  
Rotational Correlation Time ◽  
Model Systems ◽  
Quiescent Cells ◽  
Spin Resonance ◽  
In Cells

Measurements of diffusion of molecules in cells can provide information about cytoplasmic viscosity and structure. In a series of studies electron-spin resonance was used to measure the diffusion of a small spin label in the aqueous cytoplasm of mammalian cells. Translational and rotational motion were determined from the same spectra. Based on measurements made in model systems, it was hypothesized that calculations of the apparent viscosity of the cytoplasm from both rotational and translational motion would distinguish between the effects of viscosity and structure on diffusion. The diffusion constant measured in several cell lines averaged 3.3 X 10(-6) cm2/s. It was greater in growing cells and in cells treated with cytochalasin B than in quiescent cells. The viscosity of the cytoplasm calculated from the translational diffusion constant or the rotational correlation time was 2.0-3.0 centipoise, about two to three times that of the spin label in water. Therefore, over the dimensions measured by the technique, 50-100 A, solvent viscosity appears to be the major determinant of particle movement in cells under physiologic conditions. However, when cells were subjected to hypertonic conditions, the translational motion of the spin label decreased threefold, whereas the rotational motion changed by less than 20%. These data suggest that the decrease in cell volume under hypertonic conditions is accompanied by an increase in cytoplasmic barriers and a decrease in the space between existing cytoplasmic components without a significant increase in viscosity in the aqueous phase. In addition, a comparison of reported diffusion values of a variety of molecules in water and in cells indicates that cytoplasmic structure plays an important role in the diffusion of proteins such as bovine serum albumin.

scholarly journals Inositol Polyphosphate-Based Compounds as Inhibitors of Phosphoinositide 3-Kinase-Dependent Signaling

2020 ◽  
Vol 21 (19) ◽  
pp. 7198
Author(s):  
Tania Maffucci ◽  
Marco Falasca
Keyword(s):  
Therapeutic Strategy ◽  
Therapeutic Potential ◽  
Deep Understanding ◽  
Inositol Polyphosphate ◽  
Inositol Polyphosphates ◽  
Downstream Effectors ◽  
Protein Membrane ◽  
Phosphoinositide 3 Kinase ◽  
Cancer Settings ◽  
Established Role

Signaling pathways regulated by the phosphoinositide 3-kinase (PI3K) enzymes have a well-established role in cancer development and progression. Over the past 30 years, the therapeutic potential of targeting this pathway has been well recognized, and this has led to the development of a multitude of drugs, some of which have progressed into clinical trials, with few of them currently approved for use in specific cancer settings. While many inhibitors compete with ATP, hence preventing the catalytic activity of the kinases directly, a deep understanding of the mechanisms of PI3K-dependent activation of its downstream effectors led to the development of additional strategies to prevent the initiation of this signaling pathway. This review summarizes previously published studies that led to the identification of inositol polyphosphates as promising parent molecules to design novel inhibitors of PI3K-dependent signals. We focus our attention on the inhibition of protein–membrane interactions mediated by binding of pleckstrin homology domains and phosphoinositides that we proposed 20 years ago as a novel therapeutic strategy.

scholarly journals Purification and cloning of phosphatidylinositol transfer proteins from Dictyostelium discoideum: homologues of both mammalian PITPs and Saccharomyces cerevisiae Sec14p are found in the same cell

2000 ◽  
Vol 347 (3) ◽  
pp. 837-843 ◽  
Author(s):  
Philip SWIGART ◽  
Robert INSALL ◽  
Andrew WILKINS ◽  
Shamshad COCKCROFT
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Phospholipase C ◽  
Dictyostelium Discoideum ◽  
Mammalian Cells ◽  
Hl60 Cells ◽  
Single Organism ◽  
Membrane Compartment ◽  
Phosphatidylinositol Transfer ◽  
Cloned Cdna ◽  
Phosphatidylinositol Transfer Proteins

Soluble phosphatidylinositol transfer proteins (PITPs) have important roles in lipid-mediated signalling as well as in membrane traffic. Two PITPs (α and β) have been cloned from mammalian cells, which are unrelated in sequence to yeast PITP (the product of the SEC14 gene). However, all three PITPs can perform interchangeably to reconstitute function in mammalian cells. We have now purified the major PITP from the cytoplasm of Dictyostelium discoideum and cloned the gene. This protein, DdPITP1, is homologous with mammalian PITPα and PITPβ. We have also cloned a second gene (DdPITP2) related in sequence to DdPITP1. In addition, an independently cloned cDNA encodes a relative of the SEC14 family of yeast PITPs. DdPITP1, DdPITP2 and DdSec14 proteins were all able to mediate the transfer of PtdIns from one membrane compartment to another; they thus exhibited the hallmark of PITPs. Secondly, all three PITPs were able to rescue phospholipase C-mediated phosphoinositide hydrolysis in PITP-depleted HL60 cells, indicating that all three PITPs were capable of stimulating phosphoinositide synthesis. The identification of PITPs related to both mammalian PITPs and yeast Sec14p in a single organism will provide a unique opportunity to examine the functions of this class of protein with genetic approaches.

scholarly journals Cell proliferation, apoptosis and accumulation of lipid droplets in U937-1 cells incubated with eicosapentaenoic acid

1998 ◽  
Vol 336 (2) ◽  
pp. 451-459 ◽  
Author(s):  
Hanne S. FINSTAD ◽  
Christian A. DREVON ◽  
Mari Ann KULSETH ◽  
Anne V. SYNSTAD ◽  
Eirunn KNUDSEN ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Eicosapentaenoic Acid ◽  
Lipid Droplets ◽  
Lipid Peroxide ◽  
Cell Cycle Distribution ◽  
Monocytic Differentiation ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Inhibition Of Proliferation ◽  
In Cells

The monocytic cell line U937-1 was cultured in the presence of eicosapentaenoic acid (20:5, n-3) (EPA) or oleic acid (18:1, n-9) (OA). EPA caused a dose-dependent inhibition of cell proliferation, whereas OA had no effect. At the highest EPA concentrations, 120 and 240 µM, inhibition of cell proliferation was accompanied by initiation of apoptosis. A concentration of 60 µM EPA caused a 35% reduction in cell proliferation without inducing apoptosis, and was therefore used for further studies. Addition of antioxidants or inhibitors of eicosanoid synthesis had no influence on the reduced cell proliferation after EPA treatment. The inhibition required continuous presence of EPA in the incubation medium as the cells resumed a normal proliferation rate when they were placed in EPA-free medium. The inhibition of proliferation was not accompanied by differentiation into macrophage-like cells, as expression of serglycin and the ability to perform respiratory burst was unaffected by EPA. Expression of CD23 mRNA increased when the cells were incubated with EPA, but to a smaller extent than after retinoic acid (RA) or PMA treatment. Furthermore, expression of the monocytic differentiation markers CD36 and CD68 was lower in cells treated with EPA or OA when compared with untreated cells. The cell cycle distribution of U937-1 cells was similar in cells incubated with EPA or PMA, whereas RA-treated cells accumulated in the G1 phase. Side scatter increased in cells incubated with EPA and OA, which was ascribed to an accumulation of lipid droplets after examination of the cells by electron microscopy. The number of droplets per cell was higher in cells exposed to EPA than OA. The cellular triacylglycerol (TAG) increased 5.5- and 15.5-fold after incubation with OA and EPA respectively. No difference in the cellular content of cholesterol compared with untreated cells was observed. The TAG fraction in EPA-treated cells contained high amounts of EPA and docosapentaenoic acid and minor amounts of docosahexaenoic acid, whereas OA-treated cells had high levels of OA in the TAG. In cells incubated with a sulphur-substituted EPA, only minor effects on cell proliferation and no accumulation of cellular TAG were observed. These findings may indicate the existence of other mechanisms for regulation of cell behaviour by very-long-chain polyunsaturated n-3 fatty acids than the well established lipid peroxide and eicosanoid pathways.

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